The present invention relates to the synthesis of functional human hemoglobin and other proteins in erythroid tissues of transgenic non-human animals or erythroid cell lines. In addition, the present invention provides for novel expression vectors which may be used to produce xcex1-globin as well as other proteins of interest in quantity in the red blood cells of transgenic animals or erythroid cell lines; these proteins may subsequently be purified and utilized for a multitude of purposes. The human hemoglobin produced in transgenic animals according to the invention can be used as an effective, nonimmunogenic red blood cell substitute for transfusion in humans which is free of hepatitis virus and human retrovirus contamination.
Native hemoglobin exists as a tetrameric protein consisting of two xcex1 chains and two xcex2 chains. Each xcex1 and xcex2 chain binds a heme residue in noncovalent linkage. The xcex1 and xcex2 chains are also held together by noncovalent bonds resulting from hydrogen bonding and Van der Waals forces. Hemoglobin constitutes about 90% of the total protein in red blood cells, 100 ml of whole blood is capable of absorbing approximately 21 ml of gaseous oxygen.
Different molecular species of hemoglobin are produced during the embryonic, fetal, and adult life of an animal. The genes encoding the globin molecules expressed during the various developmental stages are arranged in clusters. In humans and most other mammals the xcex1 and xcex2-like gene clusters are arranged in order of their expression during development, with the embryonic genes followed by the fetal and adult globin genes (Watson et al., 1987, in xe2x80x9cMolecular Biology of the Genexe2x80x9d, Fourth Edition, The Benjamin/Cummings Publishing Co., Inc., Menlo Park, Calif., p. 650). This developmental order is not obligatory, however; for example, in the chicken, the adult xcex2-globin genes are flanked by embryonic genes.
In humans, the xcex1 globin gene cluster is located on chromosome 16 and the xcex2 globin gene cluster is located on chromosome 11. The human xcex2 globin gene cluster comprises one embryonic (xcex5), two fetal (Gxcex3 and Axcex3) and two adult xcex4 and xcex2) globin genes, which reside within approximately 50 kb of chromosomal DNA in the order 5xe2x80x2-xcex5-Gxcex3-Axcex3-xcex4-xcex2-3xe2x80x2 (Fritsch et al., 1980, Cell 19:959-972).
Expression of the human xcex2-like globin genes is precisely regulated in three important ways; they are expressed only in erythroid tissue, only during defined stages of development, and are produced at very high levels so as to rapidly establish the developmentally appropriate hemoglobin as the dominant protein in the red blood cell. The process by which the red blood cell ceases to transcribe one particular globin gene and begins to express another is referred to as xe2x80x9chemoglobin switchingxe2x80x9d. A great deal of study has been directed toward the regulatory mechanisms responsible for the switching process.
Research has indicated that DNA sequences involved in the regulation of human xcex2-globin gene expression are located both 5xe2x80x2 and 3xe2x80x2 to the translation initiation site (Wright et al., 1984, Cell 38:251-263). Analysis of constructs with xcex2-globin gene fragments inserted upstream of a reporter gene have demonstrated that sequences located immediately upstream, within, and downstream of the gene contribute to the correct temporal and tissue specific expression (Behringer et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:7056-7060; Kollias et al., 1987, Nucl. Acids Res. 15:5739-5747). Using murine erythroleukemia (MEL) and K562 cells, at least four separate regulatory elements required for appropriate expression of the human xcex2-globin gene have been identified: (i) a globin specific promoter element; (ii) a putative negative regulatory element, and (iii and iv) two downstream regulatory sequences with enhancer-like activity, one of which is located in the second intron of the xcex2-globin gene and the other located approximately 800 basepairs (bp) downstream of the gene (Behringer et al., 1987, Proc. Nat. Acad. Sci. U.S.A. 84:7056-7060). Hesse et al. (1986, Proc. Natl. Acad. Sci. U.S.A. 83:4312-4316) identified a similar enhancer sequence downstream of the chicken xcex2-globin gene in cultured chicken erythroid cells (see also Choi and Engel, 1986, Nature 323:731-734).
Active chromatin domains have been associated with overall sensitivity to DNase I digestion relative to unexpressed genes or DNA outside the active chromatin domain. Hypersensitivity sites are superimposed on the increased sensitivity of active chromatin; these DNase I hypersensitivity (HS) sites comprise approximately 100 to 200 bp of DNA which are highly susceptible to cleavage by the nuclease action of DNase I. DNase I hypersensitive sites are mapped by (i) treating nucleic acid with DNase I; (ii) isolated DNA from the nuclei; (iii) digesting the isolated DNA with a restriction enzyme; (iv) fractionating the restriction enzyme-cut DNA (i.e. by gel electrophoresis); (v) blotting the fractionated DNA on nitrocellulose; and (vi) hybridizing the nitrocellulose with a labeled probe corresponding to a subfragment of nucleic acid sequence located near the gene of interest. In addition to the full length fragment generated by the restriction enzyme, a multitude of shorter bands generated by DNase I will appear if the probe represents an area of the DNA contained in a DNase I hypersensitive site (Watson et al., 1987, in xe2x80x9cMolecular Biology of the Gene,xe2x80x9d Fourth Edition, The Benjamin/Cummings Publishing Co., Menlo Park, Calif., pp. 692-693).
Several years ago, Tuan et al. (1985, Proc. Natl. Acad. Sci. U.S.A. 82:6384-6388) and Forrester et al. (1986, Proc. Natl. Acad. Sci. U.S.A. 83:1359-1363) mapped sites that were super-hypersensitive to DNase I digestion 6-22 kilobases (kb) upstream of the xcex5-globin gene and 19 kb downstream of the xcex2-globin gene. The sites were found specifically in erythroid tissue at all stages of development. FIG. 5 depicts the location of these sites in the human xcex2-globin locus. Tuan et al. (1985, Proc. Natl. Acad. Sci. U.S.A. 82:6384-6388) observed that the major DNase I hypersensitive sites, HS I, HS II, and HS IV, situated upstream of the xcex2-globin gene, appeared to be strongly associated with xcex2-like globin gene expression since they were found to be present in K562 cells, human erythroleukemia cells, and adult human nucleated bone marrow cells (which express xcex2-like globin genes) but to be absent in HL60 cells, which do not express the xcex2-like globin genes. These experiments suggest that the super-hypersensitive sites define locus activation regions which open a large chromosomal domain for expression specifically in erythroid cells and thereby dramatically enhance globin gene expression. Furthermore, the structure of mutant loci from patients with several hemoglobinopathies suggests that the upstream hypersensitivity sites are required for efficient xcex2-globin gene expression in humans. English and Dutch xcex3xcex4xcex2-thalassemia appears to result from deletions that remove all of the upstream hypersensitivity sites (FIG. 5); although the xcex2-globin gene is intact in these patients, no xcex2-globin mRNA is produced from the mutant alleles.
The term xe2x80x9ctransgenic animalsxe2x80x9d refers to non-human animals which have incorporated a foreign gene into their genome; because this gene is present in germline tissues, it is passed from parent to offspring. Exogenous genes are introduced into single-celled embryos (Brinster et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:4438-4442). Transgenic mice have been shown to express globin (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:6376-6380), transferrin (McKnight et al., 1983, Cell 34:335-341), immunoglobulin (Brinster et al., 1983, Nature 306:332-336; Ritchie, et al., 1984, Nature 312:517-520; Goodhardt et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:4229-4233; Stall et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:3546-3550), human major histocompatibility complex class I heavy and light chain (Chamberlain et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7690-7694; functional human interleukin-2 receptors (Nishi et al., 1988, Nature 331:267-269, rat myosin light-chain 2 (Shani, 1985, Nature 314:283-286), viral oncogene (Small et al., 1985, Mol. Cell. Biol. 5:642-648), and hepatitis B virus (Chisari et al., 1985, Science 230:1157-1163) genes, to name but a few. Rearrangement of immunoglobulin genes has been observed in transgenic mice (Goodhardt et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:4229-4233; Bucchini et al., 1987, Nature 326:409-411). Krimpenfort et al. (1987, EMBO J. 6:1673-1676) generated transgenic mice that showed cell surface expression of HLA-B27 antigen biochemically indistinguishable from HLA-B27 in human cells by crossing one strain of transgenic mice carrying the HLA-B27 heavy chain gene with mice carrying the transgenic xcex22 microglobulin gene.
Correctly regulated expression of human xcex2-globin genes in transgenic nice has been observed with expression of the human gene occurring only in murine erythroid tissue (Townes et al., 1985, EMBO J. 4:1715-1723; Townes et al., 1985, Mol. Cell Biol. 5: 1977-1983). Despite inclusion of the promoter and several enhancer sequences, however, human xcex2-globin transgenes were not found to be expressed at the same levels as mouse xcex2-globin; in many cases, transgenic animals expressing the highest levels of human xcex2-globin were those which carried the greatest number of transgenes per cell. Grosveld et al. (1987, Cell 51:975-985) observed that high levels of human xcex2-globin gene expression could be obtained in transgenic animals carrying a single copy of the transgene if sequences at the extreme ends of the human xcex2-globin locus were included in the injected construction. When these sequences, which include the erythroid-specific DNase I super-hypersensitive sties, were fused upstream of the human xcex2-globin gene and injected into fertilized mouse eggs, large amounts of human xcex2-globin mRNA were synthesized and virtually all transgenic mice which developed expressed high levels of human xcex2-globin (Grosveld et al., 1987, Cell 51:975-985; Ryan et al., 1989, Genes and Development, 3:314-323; Behringer et al., 1989, Science 245:971-973; Talbot et al., 1989, Nature 338:352). Correctly regulated mouse and human xcex2-globin gene expression in cultured cells (Spandidos, et al., 1982, EMBO J. 1:15-20; Chao, et al., 1983, Cell 32:483-483; Wright et al., 1983; Nature 305:333-336) and transgenic mice (Chada, et al., 1985, Nature 314:377-380; Magram, et al., 1985, Nature 315:338-340; Townes et al., 1985, EMBO J. 4:1715-1723; Townes et al., 1985, Mol. Cell. Biol, 5:1977-1983; Costantini et al, 1985, Cold Spring Harbor Symp. Quant. Biol. 50:361-370; Kollias, et al., 1986, Cell. 46:89-94) is well documented. However, correctly regulated human xcex1-globin gene expression has been difficult to achieve. Although the xcex1-globin genes in humans are expressed exclusively in erythroid tissue, high levels of transcription from transfected xcex1-globin genes are obtained in nonerythroid culture cells (Triesman et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7428-7432; Treisman et al., 1984, Cell 38:251-263). In these same nonerythroid cells, expression of transfected xcex2-globin genes requires cis- or trans-activation by nonglobin sequences (Charney et al., 1984; Cell 38:251-263; Banerjii et al., 1981, Cell 27:299-308; Green et al., 1983, Cell 35:137-148). Transfected human xcex2-globin genes are expressed at low levels in uninduced murine erythroleukemia (MEL) cells but are transcribed at high levels when these cells are induced to differentiate (Spandidos, et al., EMBO J. 1:15-20; Chao et al., Cell 32:483-493; Wright et al., 1983, Nature (London) 305:333-336). Transfected xcex1-globin genes, on the other hand, are expressed at the same high level in uninduced and induced MEL cells (Charney et al., 1984, Cell 38:251-263). This phenomenon is observed even if the xcex1 and xcex2-globin genes are introduced into cells on the same plasmid (Charney et al., 1984, Cell 38:251-263).
Based on the results from cultured cells, xcex1-globin genes might be expected to express at high levels in transgenic mice, possibly in nonerythroid as well as in erythroid tissues. However, this has not been the case. Transgenic animals have been created that carry the human xcex11-globin gene on a 3.8-kilobase (kb) Bgl II-EcoRI fragment, the xcex12- and xcex11-globin genes in a 14-kb BAM-HI fragment, the entire human xcex1-globin locus on a 42-kb cosmid fragment, and the human xcex1- and xcex2-globin genes in various orientations on the same fragment. Although 61 animals that contain intact copies of these transgenes have been obtained, no xcex1-globin mRNA has been detected in any tissue.
Researchers have endeavored to develop transgenic animals that may be used as models for human hemoglobinopathies. Mouse models for thalassemia have been developed (xcex1-thalassemia; Martinell et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:5056-5060; xcex2-thalassemia: Skaw et al., 1983, Cell 34:1043-1052) from spontaneous mutations of the murine xcex1 and xcex2-globin genes; however, the development of animal models carrying the exact mutations found in human hemoglobinopathies, such as, for example, thalassemia, is problematic. Rubin et al. (1988, Am. J. Hum. Genet. 42:585-591 and 1988, J. Clin. Invest. 82:1129-1133) developed a strain of transgenic mice carrying the human xcex2S-globin (sickle hemoglobin) gene. Red blood cells from these mice have not been found to exhibit the sickle cell conformation; however, they have been crossed with a strain of thalassemic mice in order to study the physiology of thalassemia.
The present invention relates to the synthesis of functional human hemoglobin and other proteins in erythroid tissues of transgenic non-human animals and erythroid cell lines. It is based on the discovery that two of the five hypersensitivity sites of the xcex2-globin locus are sufficient to result in high level expression of human xcex1- or xcex2-globin transgenes.
The present invention also provides for novel recombinant nucleic acid vectors which may be used to produce xcex1-globin as well as other proteins of interest in quantity in the red blood cells of transgenic animals or cell cultures of erythroid lineage. The present invention also provides for the transgenic animals which contain these recombinant nucleic acid vectors. The vectors of the invention comprise at least one of the major DNase I hypersensitivity sites associated with the xcex2-globin locus together with a gene of interest; in preferred embodiments of the present invention, a vector comprises two of the major DNase I hypersensitivity sites associated with the xcex1-globin locus. According to various embodiments of the invention, the vectors may be used to create transgenic animals or to transfect cells in culture. In a specific embodiment of the invention, a vector which comprises two DNase I hypersensitivity sites together with the human xcex1-globin gene is used to create transgenic animals which produce human xcex1-globin protein in erythroid tissues, including red blood cells. In a preferred specific embodiment of the invention, transgenic animals are created which comprise human xcex1-globin and xcex2-globin genes, each under the transcriptional influence of two xcex2-globin locus DNase I hypersensitivity sites; these transgenic animals express human hemoglobin in their erythroid tissues, and can be used to produce human hemoglobin in quantity. In another preferred specific embodiment of the invention, transgenic animals are created which comprise the lac Z gene under the influence of xcex2-globin DNase I hypersensitivity sites, and which express the lacZ enzyme in their red blood cells.
Proteins of interest, including, but not limited to, human hemoglobin, encoded by the transgenes of the invention may be harvested in quantity from the red blood cells of transgenic animals. By including the erythroid-specific transcriptional signal in transgenes comprising a gene encoding the protein of interest, the present invention advantageously exploits the genetic programming of the red blood cell, which devotes almost its entire synthetic capabilities to the production of hemoglobin.
Human hemoglobin produced according to the methods of the present invention may be used as a red blood cell substitute in humans. Because the hemoglobin of the invention is identical to native human hemoglobin, it is nonimmunogenic; because it is produced in non-human animals it is free of such human pathogens as hepatitis virus and human retroviruses, including human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV). Production of human hemoglobin in transgenic animals offers the additional advantage of providing a red blood cell substitute which can be used to transfuse patients having any blood type whatsoever, thereby obviating the persistent problems created by limited availability of transfusable blood for rare or relatively unusual blood types.
transgenic animal: a nonhuman animal which has incorporated a foreign gene into its genome.
transgene=transgenic sequence: a foreign gene or recombinant nucleic acid construction which has been incorporated into a transgenic animal.